This invention relates to measuring the enzyme .alpha.-amylase in biological fluids. The measurement of .alpha.-amylase in urine and serum is widely performed in the diagnosis of pancreatic disorders. A number of assay described in the literature employ oligosaccharide .alpha.-amylase substrates, which are cleaved into smaller chains by .alpha.-amylase (which is an endo-enzyme), in conjunction with an exo-enzyme, e.g., .alpha.-glucosidase, .beta.-glucosidase, or glucoamylase.
Driscoll et al. U.S. Pat. No. 4,102,747 describes an assay employing oligosaccharides of chain length 4-10 glucose units, with a chromophore (p-nitrophenol, or "pNP") on the reducing end. The chain is "resistant to cleavage by .alpha.-glucosidase", and cleavage by .alpha.-amylase produces "smaller fragments which are acted upon by .alpha.glycosidase . . . to liberate p-nitrophenol."
Marshall et al. (1977) Clin Chimica Acta 277 describes an assay employing "modified amylaceous polysaccharides containing blockages to the action of glucoamylase. Such blockages to exo-enzyme action are conveniently introduced . . . by limited periodate oxidation . . . or by substitution of monosaccharide residues." "In the presence of excess glucoamylase, the amount of glucose released . . . is directly proportional to the amount of .alpha. amylase present." "Glucose was determined by the glucose oxidase method."
A brochure published by Calbiochem-Behring describes an assay, the "Pantrak.RTM. E. K. Amylase" method, similar to that of Driscoll et al., supra.
A biomedix.RTM. catalog describes the DeltaTest.RTM. Assay, similar to that of Marshall et al., supra.
Three patents assigned to E. I. DuPont de Nemours and Company (Burns et al. U.S. Pat. No. 4,145,527; Farnham et al. U.S. Pat. No. 4,147,860; and Menson et al. U.S. Pat. No. 4,233,403) describe assays similar to that of Driscoll et al., supra.
U.K. Pat. Appln. GB 2004646 describes an assay employing maltoheptaose as the .alpha.-amylase substrate.